DNA helicases are ATP-dependent motor proteins that unwind duplex DNA to form the single stranded (ss) DNA intermediates required for DNA metabolism in all organisms. We are studying the kinetic mechanisms of DNA unwinding and DNA translocation of three non-hexameric SF1 DNA helicases from E. coli, Rep, UvrD, and RecBCD, which function in replication, repair, and recombination, respectively. RecBCD is a hetero-trimeric complex containing two SF1 helicases (B and D). Our pre-steady state kinetic studies indicate that Rep and UvrD helicases function as oligomers in vitro, even though monomers of these enzymes can translocate efficiently along ss-DNA. We have discovered an important regulatory domain within these monomers (2B domain) that, when removed, activates helicase activity of a Rep monomer in vitro. Although the 2B domain is not needed for translocation, large movements of the 2B domain are coupled to DNA binding and ATP hydrolysis, hence we will study the role of the 2B domain in monomer ssDNA translocation and DNA unwinding by oligomers. We will use mutagenesis and transient kinetic approaches (stopped-flow and chemical quenched-flow) and methods of analysis that we have developed to examine the details of how the monomeric molecular motor functions in translocation. We will also test current hypothesis for how the oligomeric helicase complexes unwind DNA, with the goal of developing a full kinetic mechanism for unwinding. SF1 helicases also function to disrupt protein-DNA complexes, and we will study the mechanism by which UvrD disrupts RecA-ssDNA filaments. DNA binding, ATP hydrolysis and DNA unwinding by RecBCD and RecBC helicases will also be examined mechanistically. Thermodynamic and kinetic studies will be used to understand how these proteins destabilize (melt) DNA base pairs in a Mg2+dependent, but ATP-independent reaction. We will also examine the mechanism by which accessory proteins, MutL for UvrD and PriC for Rep, stimulate DNA unwinding. Our ensemble studies will be complemented by single molecule studies of DNA binding, translocation and unwinding by these helicases. The overall goal is to obtain a molecular understanding of the kinetic mechanism(s) by which these molecular motors translocate along and unwind DNA and how these processes are coupled to ATP binding and hydrolysis. PUBLIC HEALTH RELEVANCE: DNA helicases and translocases play fundamental roles in all aspects of DNA metabolism, including DNA replication, recombination and repair in all organisms including humans. Mutations in a number of human DNA helicases are linked to several human diseases, including Werner's and Bloom's syndromes. Because of their pivotal roles in nucleic acid metabolism, these enzymes are prime targets for drugs that may inhibit them specifically, and it is critical to understand their mechanisms of action. [unreadable] [unreadable] [unreadable]